Tuber Protein Research Articles

Effect of light quality on growth and quality of Pinellia ternata

Measuring the content of soluble reducing sugar, total sugar, soluble protein, guanosine, alkaloids, and succinic acid of Pinellia ternata tuber were measured by anthrone-sulfuric acid colorimetric method, Coomassie brilliant blue method, RP-HPLC, reverse potentiometric titration, acid dye colorimetry, respectively. The result showed that yellow light could promote the growth and development of P. ternata and increase the content of soluble reducing sugar, total sugar, alkaloids, and succinic acid. Under blue light could promote the content of soluble protein and guanosine. Red and yellow light increased the content of chlorophyll a and chlorophyll b, contrastively blue light reduced the content of chlorophyll a and chlorophyll b. White film through the most uniform spectrum was most conducive to the synthesis of chlorophyll a. As single film, blue film, yellow film were more conducive to the synthesis of chlorophyll a, green film and red film had been relatively beneficial to the synthesis of chlorophyll b. Bulbil formed the largest number and the biggest propagation coefficient of P. ternata under red light showed that it could increase the production of P. ternata under red light.

Relationship Between Tuber Storage Proteins and Tuber Powdery Scab Resistance in Potato

The potato genotypes (Solanum tuberosum L.) with russet tuber skin are generally resistant to powdery scab (Spongospora subterranea f.sp. subterranea or Sss). Lipoxygenase (LOX; EC 1.13.11.12) and patatin are two key storage proteins that are known to offer resistance to several diseases and insects. The objective of this study was to find out the relationship of these proteins in stored tubers with potato tuber powdery scab resistance, especially in russet skinned potatoes. An evaluation of potato germplasm with different tuber characteristics in a greenhouse environment over several years (2006–’11) suggests that russet skinned tuber genotypes (Mesa Russet, Centennial Russet and Russet Nugget) with negligible tuber disease severity index (DSI) and 100 % marketability were resistant to powdery scab. Higher physiological levels of LOX protein (on a dry weight basis) were negatively correlated with tuber DSI and positively correlated with tuber russet skin. Tuber total protein and patatin-lipase levels did not have a significant relationship with tuber powdery scab resistance. The proposed role of LOX protein in suberin- and/or non-suberin-mediated mechanisms of powdery scab resistance in russet skinned tubers are discussed here. The physiological levels of LOX protein can be considered as a useful marker for powdery scab resistance in potato breeding programs.

Accumulation of PrLeg, a perilla legumin protein in potato tuber results in enhanced level of sulphur-containing amino acids

Potato is the fourth staple food in the world, following rice, wheat, and maize, whereas tubers contain high quality of starch, relatively high amounts of vitamin C and many other important substances. It also contains relatively good quality of protein (about 3 to 6% of the dried weight) and patatin, and 11S globulin is a major storage protein with high level of lysine. However, tuber protein contains relatively low amounts of sulphur-containing amino acids, which may result in low nutritional value. Recently, we cloned a gene encoding PrLeg polypeptide, a seed storage protein from perilla, which contains relatively higher levels of sulphur-containing amino acids. We transformed PrLeg cDNA into a potato plant to over-express under the direction of the tuber-specific promoter, patatin. Most of the transgenic lines identified through PCR and RT–PCR analyses were able to accumulate high amount of prLeg transcript in their tuber tissue, while very little or no transcript that were detected in their leaf tissues. The level of methionine content was elevated up to three-fold compared to non-transgenic parental line, without any significant changes in other amino acids, suggesting that further research is required to get a deeper insight into their nutritional value.

Variation of nitrogenous components in potato (Solanum tuberosum L.) tubers produced under organic and conventional crop management

The information related to protein and other nitrogenous components in organically produced potato tubers can be interesting for consumers with specific diets. Such information is still limited. Here we report results on the effects of organic and conventional production systems on accumulation of nitrogenous substances including total tuber protein and the nutritiously most important tuber protein, patatin, observed in five cultivars on two localities during three consecutive years. Organically produced potato tubers contained significantly lower content of total nitrogen, crude protein, nitrates and glycoalkaloids (α-solanine and α-chaconine). Protein content, patatin relative abundance in total tuber protein and patatin content in organically produced potato tubers were slightly lower than in conventional ones but the differences were not statistically significant. The cultivar and interactions between cultivars and other production factors affected considerably contents of crude protein, protein, glycoalkaloids and patatin relative abundance. The effect of cultivar on these nitrogenous characteristics was stronger for potatoes produced under conventional farming indicating the sensitive reaction of organically produced potatoes on less favourable agro-ecological production conditions. The results indicate that cultivar-specific genetic factors play the predominant role in the reciprocal interaction with other cultivation factors affecting the nitrogenous qualitative parameters of organically produced potato tubers. This should be considered to include primarily the cultivar in quality standards of organic farming. The late maturing cultivar Bionta exhibited high instability in tubers yield, total protein and patatin protein characteristics when produced under organic farming system, while the early maturing cultivar Karin reacted on the conditions of organic farming by an increase of the patatin content.

Different Nitrogen Sources Affect Biomass Partitioning and Quality of Potato Production in a Hydroponic System

Crop production systems should reduce nitrogen application costs and assure that the appropriate form of nitrogen is used. Thus, three potato cultivars in a hydroponic system were supplied with two different nitrogen sources to determine the effect on biomass accumulation and partitioning, and total plant nitrogen content. Cultivars Agata, Atlantic and Bintje received, on alternate days, nutritive solutions differing only in either calcium nitrate or urea. Urea stimulated biomass accumulation and total nitrogen in shoots. Moreover, urea reduced the ratios tuber:shoot and tuber:root, and increased the ratio shoot:root, indicating competition for biomass partitioning between shoots and tubers. Urea stimulated greater tuber volume in the Atlantic cultivar, and increased tuber protein content, which is undesirable for industry. These results suggested that salts containing nitrate could be more appropriate for hydroponic potato cultivation, since urea compromised tuber quality and biomass partitioning in all cultivars studied.

Effects of Leonardite Applications on Yield and Some Quality Parameters of Potatoes (Solanum tuberosum L.)

The present study was carried out at Suleyman Demirel University research farm in Isparta during2008 and 2009 crop seasons to determine the effects of leonardite (54.5% organic matter) on yield and quality of potato. Four leonardite doses (0, 200, 400, 600 kg ha-1) and four potato cultivars (Van Gogh, Milva, Lady Olympia, Agata) were used in the study. A total of seven traits (plant height, tuber number per plant, marketable tuber yield, total tuber yield, protein content, vitamin C content and specific gravity) were measured. Significant differences were detected between control and leonardite applications for all the traits studied. Statisticallysignificant differences were not found between the leonardite doses for plant height and specific gravity. There was no significant difference between 400 and 600 kg ha-1 leornardite application for tuber number per plant, marketable tuber and total tuber yield, protein and vitamin C contents. Leonardite applications increased number of tubers per plant by 22%, marketable tuber yield by 38% and total tuber yield by 15% compared with the control. These results suggest that 400 kg ha-1 leonardite combination to the standard fertilization will be sufficient to obtain adequate yield and quality tubers in potatoes

Expression of an engineered granule-boundEscherichia coliglycogen branching enzyme in potato results in severe morphological changes in starch granules

The Escherichia coli glycogen branching enzyme (GLGB) was fused to either the C- or N-terminus of a starch-binding domain (SBD) and expressed in two potato genetic backgrounds: the amylose-free mutant (amf) and an amylose-containing line (Kardal). Regardless of background or construct used, a large amount of GLGB/SBD fusion protein was accumulated inside the starch granules, however, without an increase in branching. The presence of GLGB/SBD fusion proteins resulted in altered morphology of the starch granules in both genetic backgrounds. In the amf genetic background, the starch granules showed both amalgamated granules and porous starch granules, whereas in Kardal background, the starch granules showed an irregular rough surface. The altered starch granules in both amf and Kardal backgrounds were visible from the initial stage of potato tuber development. High-throughput transcriptomic analysis showed that expression of GLGB/SBD fusion protein in potato tubers did not affect the expression level of most genes directly involved in the starch biosynthesis except for the up-regulation of a beta-amylase gene in Kardal background. The beta-amylase protein could be responsible for the degradation of the extra branches potentially introduced by GLGB.

Sequential Extraction Results in Improved Proteome Profiling of Medicinal Plant Pinellia ternata Tubers, Which Contain Large Amounts of High-Abundance Proteins

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.

Molecular cloning and sequence comparison of a cDNA encoding α-glucan, water dikinase (GWD) from potato (Solanum tuberosum L.), and analysis of gene expression

Starch phosphorylation is an important biochemical aspect of plant starch metabolism as it influences the overall structure of the starch granule, and a prerequisite for its degradation. There is a growing interest on the isolation and characterization of α-glucan/glucan-like, water dikinases (GWDs) from plants, particularly agriculturally important crops, because GWD is known to catalyze starch phosphorylation both in leaves and different plant storage organs. In the present study, a 4,789-bp full-length cDNA encoding a GWD isoform was isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. The predicted protein consisted of 1,463 amino acids having N-terminal 77-amino acid transit peptide, and 1,386-amino acid mature protein shorter by one amino acid as compared to the other mature GWDs from potato and tomato. The mature GWD showed 98 % sequence identity with the GWD isolated earlier from the potato cv. Desiree. Variations were found at 25 locations representing mostly non-conservative substitutions. The GWD represents a distinct isoform from potato, as revealed by sequence and phylogenetic analyses. Amino acid composition, segment-wise hydrophobic characters, predicted secondary structures were also analyzed and documented in this report. Broadly, the level of GWD expression as analyzed by semi-quantitative RT-PCR approach was found to be nearly uniform both in the mature tubers and leaves from most of the potato cultivars. By immunodetection technique, a band corresponding to ~155 kDa protein was detected only in the tuber protein extracts. The tuber starch-bound phosphorus content data showed minor variations between the potato cultivars.

Concentrations of some macroelements in potato tubers stored at 4ºC and 8ºC

The objective of the investigations was to evaluate changes in concentrations of total protein (nitrogen x 6.25), phosphorus and potassium in tubers of two potato cultivars: Rywal and Saturna, as dependent on the application of herbicides (Afalon 50 WP, Azogard 50 WP, Sencor 70 WG, and Apyros 75 WG), after harvest and after 3 and 6 months of storage. Concentrations of total protein, phosphorus and potassium in potato tubers were strongly conditioned by genetic features of the cultivars. The tubers collected from the objects sprayed with the herbicides over the vegetation period contained by 3.7%, 8.1%, and by 3.5% more protein, phosphorus and potassium, respectively, than those nursed exclusively mechanically. The corresponding values in the latter case reached respectively (means for the objects with the use of herbicides) 24.4 g kg-1, 660.5 mg kg-1 and 5351.3 mg kg-1. After 3 and 6 months of storage at 8°C, the content of nitrogen was significantly lower. Similarly, tubers stored at 4oC contained significantly less nitrogen, but not earlier than after 6 months. In contrast, concentrations of phosphorus and potassium did not change significantly over the storage of tubers in chambers with the lower temperature (4°C).

Molecular cloning, structural analysis and mass spectrometric identification of native dioscorins of various yam species

Dioscorins are the major storage proteins of yam tubers. However, the molecular nature of their heterogeneity in tubers has not been fully elucidated. In this study the authors isolated the dioscorin gene families of Dioscorea japonica and Dioscorea pseudojaponica, performed matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and elucidated which dioscorin isoforms are the major constituents in tubers. The dioscorin gene families of D. japonica (Dj-dioA1-Dj-dioA4, Dj-dioB1 and Dj-dioB2) and D. pseudojaponica (Dp-dioA1-Dp-dioA5 and Dp-dioB1) were cloned from cDNA libraries of yam tubers. The dioscorins isolated from Dioscorea alata (Da-dioscorins), D. japonica (Dj-dioscorins) and D. pseudojaponica (Dp-dioscorins) were mainly monomers, with a few dimers. The monomers contained one intramolecular disulfide bond (Cys(28)-Cys(187)) and belonged to Class A dioscorins with two cysteine residues. The dimers consisted of Class B dioscorins with one intermolecular disulfide bond (Cys(40)-Cys(40)). Results of MALDI-TOF-MS revealed that the Da-dioscorins were mainly encoded by Da-dioA2, Da-dioA3 and Da-dioA4. The majority of the Dj-dioscorins were encoded by Dj-dioA1, Dj-dioA2, Dj-dioA3 and Dj-dioB2. The Dp-dioscorins mainly comprised proteins encoded by Dp-dioA1, Dp-dioA3, Dp-dioA4, Dp-dioB1 and Dp-dioB2. Determination of the constituents of dioscorin isoforms in yam tubers provides a basis for future studies of their physiological and biomedical functions.

Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities

Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15mg proteins per liter Escherichia coli culture (mg/L) to 30mg/L of rDj-dioscorinA3 and 4 to 8mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys28 and Cys187 residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins.

Structure of a post-translationally processed heterodimeric double-headed Kunitz-type serine protease inhibitor from potato

Potato serine protease inhibitor (PSPI) constitutes about 22% of the total amount of proteins in potato tubers (cv. Elkana), making it the most abundant protease inhibitor in the plant. PSPI is a heterodimeric double-headed Kunitz-type serine protease inhibitor that can tightly and simultaneously bind two serine proteases by mimicking the substrate of the enzyme with its reactive-site loops. Here, the crystal structure of PSPI is reported, representing the first heterodimeric double-headed Kunitz-type serine protease inhibitor structure to be determined. PSPI has a β-trefoil fold and, based on the structure, two reactive-site loops bearing residues Phe75 and Lys95 were identified.

Tuber quality assessment of orange-fleshed sweet potato (Ipomoea batatas) cultivars and their genetic relatedness as revealed by SDS-PAGE of tuber proteins

Orange-fleshed sweet potato (OFSP) is an important source of β-carotene and can make a major contribution in alleviating vitamin A malnutrition. Replacement of white-fleshed varieties with new b-carotene-rich OFSP cultivars that meet local preference would benefit the children who are at risk for vitamin A deficiency related problems. Twelve OFSP cultivars of diverse origin were analyzed for biochemical and antioxidant composition, viz dry matter, crude protein, crude fat crude fibre, carbohydrate, starch, amylose, reducing sugars, total phenols, ascorbic acid and b-carotene contents of their tubers. Many of the investigated cultivars have high levels of dry matter, crude protein, starch, β-carotene, phenols and ascorbic acid. Cultivars with high dry matter and b-carotene can be utilized to develop cultivars for commercial exploitation. This paper also highlights the genetic relatedness among the OFSP cultivars, assessed by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of tuber proteins. High genetic similarities were observed among the cultivars originated in India.

Increased expression of matrix metalloproteinase 9 in cortical lesions from patients with focal cortical dysplasia type IIb and tuberous sclerosis complex

The malformative cortical lesions in the cerebral cortex that are characteristic of focal cortical dysplasia type IIb (FCDIIb) and tuberous sclerosis complex (TSC) are well-recognized causes of chronic intractable epilepsy in children. Increasing evidence suggests that extracellular matrix molecules play important roles in epileptogenesis. Matrix metalloproteinase 9 (MMP9), a typical extracellular matrix proteolytic protease, has been shown to participate in the occurrence of seizures in experimental models. In the present study, we used immunoblotting to analyze the levels of MMP9 protein in FCDIIb lesions, TSC tubers and control samples, which included epileptic neocortices from temporal lobe epilepsy and non-epileptic normal cortices (CTX). The cellular distribution of MMP9 was further investigated by immunohistochemical methods. Our findings demonstrated the elevated levels of the inactive and active forms of MMP9 protein in FCDIIb and TSC lesions compared with CTX. Furthermore, the immunohistochemical results showed that MMP9 was characteristically expressed in the following misshapen cells: hypertrophic neurons, dysmorphic neurons, balloon cells and giant cells. Additionally, double immunofluorescent staining revealed that the reactive astrocytes, but not the microglia, expressed high levels of MMP9. Taken together, our findings suggest that the overexpression and spatial distribution patterns of MMP9 may be linked with the intractable epilepsy caused by FCDIIb and TSC.

Soil nitrogen variability in relation to seasonal nitrogen cycling and accumulation of nitrogenous components in starch processing potatoes

Seasonal dynamics of total soil nitrogen, ammonium and nitrate ions contents were evaluated for a processing potato field experiment conducted on three sites with different altitudes. Fertilization levels of 100 and 200 kg N ha−1 applied before potato crop planting were compared. Content of soil ammonium and nitrate ions ranged from 1.5 to 149 mg kg−1 and from 3.0 to 108 mg kg−1 on soil dry matter basis, respectively and showed strong seasonal dynamics with a rapid increase of the content during June and subsequent fall down to initial values. The nitrogen fertilization level had minimal effect on seasonal dynamic of total soil nitrogen, ammonium and nitrate ions contents. The higher fertilization level significantly increased crude protein content in potato tubers and yield of crude protein per area unit. The factors of starch content in potato tubers and yield per area unit were affected negatively by the higher dose of nitrogen fertilization. It could be concluded that the dose of 100 kg N ha−1 is sufficient for production of potato tubers with high content of both crude protein and starch, especially in production areas with higher altitude. The dose of 200 N is justifiable only in production areas with lower altitude (below 400 m a.s.l.) that are not so suitable for accumulation of crude protein and starch in potato tubers.

Analysis of Natural Variation of the Potato Tuber Proteome Reveals Novel Candidate Genes for Tuber Bruising

Potato (Solanum tuberosum) presents a challenging organism for the genetic and molecular dissection of complex traits due to its tetraploidy and high heterozygosity. One such complex trait of high agronomic interest is the tuber susceptibility to bruising upon mechanical impact, which involves an enzymatic browning reaction. We have compared the tuber proteome of two groups of 10 potato cultivars differing in bruising susceptibility to (i) identify de novo proteins that contribute to bruising, based on differential protein expression, and (ii) validate these proteins by combining proteomics with association genetics. The comparison of 20 potato varieties yields insight into the high natural variation of tuber protein patterns due to genetic background. Seven genes or gene families were found that were both differentially expressed on the protein level between groups and for which DNA polymorphisms were associated with the investigated traits. A putative class III lipase was identified as a novel factor contributing to the natural variation of bruising. Additionally, tuber proteome changes triggered by mechanical impact, within and between groups, were monitored over time. Differentially expressed proteins were found, notably lipases, patatins, and annexins, showing remarkable time-dependent protein variation.

CONSERVATION OF POTATO GERMPLASM IN THE CZECH REPUBLIC

In this study we tested a new cryopreservation method for conservation of old potato cultivars of the Czech origin. Explants recovered after cryopreservation were introduced into ex vitro conditions and tests of genotype stability were evaluated by molecular markers, morphological traits and tuber proteins. Fifty old potato cultivars of the Czech origin were cryopreserved and successfully recovered. Genetic stability of cryopreserved cultivars was evaluated in 29 cultivars by molecular markers, 14 cultivars by morphological traits and 8 cultivars by tuber protein analyses. Plants regenerated after cryopreservation were compared with plants from in vitro bank. No significant differences were found between the samples before and after cryopreservation. The results showed the possibility of using the cryopreservation method for long-term storage of potato germplasm.

The development of a high-yield recombinant protein bioreactor through RNAi induced knockdown of ATP/ADP transporter in Solanum tuberosum

There is an increased need for high-yield protein production platforms to meet growing demand. Tuber-based production in Solanum tuberosum offers several advantages, including high biomass yield, although protein concentration is typically low. In this work, we investigated the question whether minor interruption of starch biosynthesis can have a positive effect on tuber protein content and/or tuber biomass, as previous work suggested that partial obstruction of starch synthesis had variable effects on tuber yield. To this end, we used a RNAi approach to knock down ATP/ADP transporter and obtained a large number of transgenic lines for screening of lines with improved tuber protein content and/or tuber biomass. The initial screening was based on tuber biomass because of its relative simplicity. We identified a line, riAATP1-10, with minor (less than 15%) reduction in starch, that had a nearly 30% increase in biomass compared to wild-type, producing both more and larger tubers with altered morphological features compared to wild-type. riAATP1-10 tubers have a higher concentration of soluble protein compared to wild-type tubers, with nearly 50% more soluble protein. We assessed the suitability of this line as a new bioreactor by expressing a human scFv, reaching over 0.5% of total soluble protein, a 2-fold increase over the highest accumulating line in a wild-type background. Together with increased biomass and increased levels in total protein content, foreign protein expression in riAATP1-10 line would translate into a nearly 4-fold increase in recombinant protein yield per plant. Our results indicate that riAATP1-10 line provides an improved expression system for production of foreign proteins.

Systemin, Hydroxyproline-Rich Systemin and the Induction of Protease Inhibitors

Systemin, an 18-amino acid signaling peptide isolated from tomato leaves, has been found to be an integral component of the jasmine acid signaling pathway, leading to the synthesis of protease inhibitors (PIs). The discovery of systemin has led to a search for other peptide signals involved in defense in the Solanaceae and in other plant families. A new class of peptides having similar signaling properties but little sequence homology to systemin have been found and termed hydroxyproline-rich glycopeptide systemins (HypSys). These small (18-20 amino acids) glycopeptides, like systemin, are derived from larger precursor proteins (proHypSys) and until recently were thought to function only in protection from herbivore attack. However, HypSys peptides isolated from petunia induced the defensin gene, known for its involvement in pathogen defense. More recently, a HypSys glycopeptide was isolated from sweet potato, a member of the Convolvulaceae family and found to induce the sporamin gene which codes for the major storage protein in tubers with trypsin inhibitor activity. These recent discoveries expand the function and range of the HypSys family of glycopeptides and establish these unique inducible signaling molecules as potential components of defense pathways throughout the Eudicots. Herein we review the signaling and structural properties of systemin and the HypSys glycopeptides and their roles in the induction of PIs.